Genistein induces long-term expression of progesterone receptor regardless of estrogen receptor status and improves the prognosis of endometrial cancer patients

Progesterone is used to treat uterine endometrial cancer in young patients wishing to preserve their fertility as well as in advanced or recurrent patients, but its response rate is limited. The antitumor effect of progesterone is mediated by progesterone receptor (PR) binding. Hence, loss of progesterone’s therapeutic effect, i.e., development of progesterone resistance, is mainly due to decreased PR expression. However, little is known about underlying mechanisms that regulate PR expression. Immunohistochemistry analysis of specimens from 31 young, endometrial cancer patients showed that elevated PR expression significantly increased (P < 0.05) rates of progression-free and overall survival. We investigated mechanisms of regulating PR expression and suppressing cell proliferation using genistein, a chemotherapeutic agent against different cancers. Genistein inhibits cell growth by inducing cell cycle arrest in G2 and apoptosis; moreover, it upregulates prolonged expression of PR-B and forkhead box protein O1, regardless of estrogen receptor alpha expression in endometrial cancer cells. Genistein-induced PR expression decreases CCAAT/enhancer binding protein beta expression and activates c-Jun N-terminal kinase pathway, rather than causing epigenetic alterations of the PR promoter. Therefore, increased PR expression is an important antitumor effect of genistein. This may help to improve the response rates of fertility-sparing treatments for young patients.

www.nature.com/scientificreports/ transactivation function (AF3) region 6 . Several reports have shown that the functional activities of PR-A and PR-B differ in a cell type-, promoter-, or ligand-specific manner [7][8][9] . Hence, it is possible that the different mechanisms for regulating the PR isoforms are reflected through the differential expression of the PRs in the endometrium. It has been hypothesized that two promoters, namely Promoter A (+ 464 to + 1105) and Promoter B (− 711 to + 31), are responsible for the production of PR-A and PR-B, respectively. Interestingly, neither PR-A nor PR-B contains a palindromic estrogen response element (ERE). A previous report had proposed that the estrogen responsiveness of the human PR gene is derived in part from the interaction of estrogen receptor α (ERα) and specificity protein 1 (Sp1) with a region in Promoter A lying between + 571 and + 595; this site contains a half ERE site and two adjacent Sp1 sites, and it is referred to as the + 571 ERE/Sp1 site 10 . In contrast, a large proportion of in vitro evidence suggests that the absence or reduced expression of PR-B, but not PR-A, might result in the failure of progesterone treatment; this is called progesterone resistance, and it leads to aberrant PR-B-mediated signaling in endometrial cancer cells 11 . More than 30% of patients with estrogen-dependent and well-differentiated endometrial cancer fail to respond to progesterone treatment due to this progesterone resistance 12 . Hence, to overcome this, it is essential to clarify the underlying mechanisms of progesterone resistance. Genistein is a biologically active isoflavone found in soy products. It has been shown to modulate several pathophysiological pathways that are commonly deregulated in obesity, metabolic syndromes, and cancer. The structural similarities between genistein and estradiol can also elucidate its potential role in treating postmenopausal symptoms, such as reduction in bone mass and hot flushes 13 . Genistein also has an anti-proliferative effect on various cancer cells, including endometrial cancer 14 . There are a few reports on the action of genistein on hormone receptors in endometrial cancer cells; however, one study has indicated that genistein decreases ERα mRNA expression while increasing PR expression 15 . Therefore, we examined the genistein-induced expression of each PR isoform and clarified its role in PR regulation as well as its PR-mediated antitumor effects.

Results
Expression of PR in endometrial cancer is linked to prognosis in young patients. First, we performed immunohistochemical analysis of tissue specimens collected from endometrial cancer patients aged < 40 years. Patient characteristics are shown in Table 1. Of the 32 patients' specimens, 31 were available for analysis. The H-score distribution of PR is presented in Table 2. Incidentally, the H-score of PR is significantly associated with the clinical stage (between FIGO stage I or II and III or IV, P < 0.01) of the disease. Moreover, it is significantly related to myometrial invasion (P < 0.05 between cases with no invasion and those with > 50% myometrial invasion, and P < 0.01 between cases with < 50% myometrial invasion and those with > 50% myometrial invasion) (Fig. 1A, B). However, there are no significant associations between PR expression and the histological type or lymph node metastasis (Fig. 1C, D). The Kaplan-Meier analysis indicated that the level of PR expression is correlated with progression-free survival (P < 0.05, Fig. 1E) and overall survival (P < 0.05, Fig. 1F) when the cases were divided into two groups by PR-H-score 100 (Fig. 1G), thereby suggesting that PR expression could be an independent prognostic factor for endometrial cancer. www.nature.com/scientificreports/  ERα-dependent Ishikawa and ERα-independent KLE, we performed the water-soluble tetrazolium salt-8 (WST-8) assay. Interestingly, genistein significantly inhibits cell growth in a dose-and time-dependent manner in both the cell lines ( Fig. 2A). Subsequently, in order to evaluate the underlying mechanism of growth inhibition by genistein, we performed flow cytometry analysis. Ishikawa and KLE cells were treated with 20 μM genistein for 6 days, and it caused the accumulation of cells in the G2/M-and sub-G1-phases ( Fig. 2B, C). Our western blot analysis also showed a decrease in phospho-histone H3 (Ser10), a representative marker of mitotic phase, and an increase in cdc2 phosphorylation after 48 h of exposure to genistein in both the cells; however, there was no change in cdc25 phosphorylation (Fig. 2D). We also detected an increase in cleaved caspase-3, thereby indicating the initiation of apoptosis. These results indicate that genistein induces cell cycle arrest at the G2/M phase in endometrial cancer cells, followed by their apoptosis.
Genistein upregulates the expression of PR and forkhead box protein O1 (FOXO1) regardless of ERα expression in endometrial cancer cells. Next, we performed quantitative polymerase chain reaction (qPCR) assays and western blot analyses to elucidate the effect of genistein on PR expression in endometrial cancer cell lines. For that, we first confirmed that Ishikawa cells show higher expression of ERα and lower expression of PR-B, as compared to that in KLE cells ( Supplementary Fig. 1). There is no significant difference in ERß expression between the two cells, and PR-A expression is low in both the cell lines. We evaluated the differences in ER and PR expression owing to the effect of the specific isoflavone treatment, namely genistein, daidzein, glycitein, and equol. The expression of ERα, ERß, PR-AB, and PR-B was examined by qPCR in Ishikawa and KLE cells treated with each of the isoflavone metabolites (20 μM) for 6 days. We observed that genistein upregulates PR-AB expression to the highest level, as compared to the effect of other isoflavone metabolites, without increasing the expressions of ERα and ERß in both the cancer cell lines (Fig. 3A).
To elucidate the effect of genistein on ER and PR expressions over a short period without medium replacement, both the receptor expressions were assessed after 0, 3, 6, 24, 48 and 72 h of treatment with 20 μM genistein. We observed that genistein significantly induces the expression of PR-B in Ishikawa cells and that of PR-AB in Ishikawa and KLE cells after 48 h, but it does not change the expressions of ERα and ERß in either of the cell lines ( Fig. 3B, C). Similarly, the effect of genistein over a long period with replacement of the medium every 2 or 3 days was examined every day for 6 days in the Ishikawa cells and every 1 or 2 days for 9 days in the KLE cells. We observed that the effects of genistein on increasing PR-AB and PR-B expressions are retained until the final time point in both the cell lines (Fig. 3D, E). To further confirm this observation, we used western blot analyses to assess the expressions of ER, PR, FOXO1, a tumor suppressor located downstream of PR signaling, and insulin-like growth factor-binding protein 1 (IGFBP1), a factor associated with the decidua. Interestingly, www.nature.com/scientificreports/ Effect of genistein on tumor growth in a mouse xenograft model. To further evaluate the effect of genistein on endometrial cancer cells in vivo, athymic nude mice were subcutaneously inoculated with Ishikawa cells. We observed that genistein significantly suppresses tumor growth, as compared to the control group, without reducing body weight (Fig. 5A, B). Thereafter, we analyzed post-treatment apoptosis in the tumors using the TUNEL assay. There was no difference in apoptosis between the genistein-treated and control groups (Supplementary Fig. 8). The tissue sections were also immunohistochemically stained with Ki-67, a representative cell proliferation marker. Genistein treatment significantly reduces the percentage of Ki-67-positive cells (Fig. 5C). Additionally, we assessed the effect of genistein on ERα and FOXO1 expressions in vivo using immunostaining. Genistein treatment significantly reduces the expression of ERα in the tumors (Fig. 5D).

Discussion
We revealed that PR expression is associated with favorable clinical outcomes in young patients with endometrial cancer who wish to preserve their fertility. Moreover, we elucidated the ER-independent inhibition of cell proliferation as well as the mechanism of increased PR expression in endometrial cancer using genistein. In previous studies using qPCR and immunostaining, there have been various reports on PR isoform status related to prognosis, namely PR-A and/or PR-B [18][19][20][21] . Therefore, it is natural that an absence of the PR status is associated with poor prognosis. Our study obtained similar results, even though it was limited to endometrial cancer in young patients aged < 40 years, thereby suggesting that increased PR expression leads to improved survival, regardless of progestin therapy. These findings are important for the development of treatment options for patients with endometrial cancer who desire to preserve their fertility. First, our results demonstrated that genistein suppresses cell proliferation by inducing cell cycle arrest at the G2-M phase as well as apoptosis, and it upregulates the expression of FOXO1. Although our study did not find a convincing apoptotic effect of genistein via the TUNEL assay, a previous study has reported that it increased the expression of Bax, Bad, and Bak in vivo which promoted tumor apoptosis 22 . The discrepancy in these findings may be related to the use of different analytical methods for the detection of apoptosis. Many previous studies have reported that genistein causes G2/M block in cell cycle progression and suppresses cell proliferation in vitro and in vivo not only in endometrial cancer, but also in cases of breast cancer, prostate cancer, hepatic cancer, and bladder cancer 15,[22][23][24][25][26][27][28] . However, the concentration of genistein has opposite effects on cell growth in breast cancer 29 . Particularly, the anti-proliferative effects of genistein that are mainly observed at high treatment doses (> 20 μM) are independent of ER, while the proliferative effects observed at low treatment doses (0.01-10 μM) are ER-mediated [30][31][32][33][34] . In ER-negative cells, this dual effect is not observed, and genistein produces only antiproliferative effects, especially at high doses 35,36 . Similarly, in our study, ER-positive Ishikawa cells exhibited a low cell growth after 72 and 96 h of genistein exposure, while ER-negative KLE cells did not show this change. The FOXO family, including FOXO1, is a key effector of phosphoinositide 3-kinase (PI3K) deregulation since it is a direct downstream phosphorylation target of the protein kinase PKB and the related kinase SGK1 37  www.nature.com/scientificreports/ of the PI3K/PKB signaling pathway 39 . Incidentally, FOXO1 is upregulated by progestin in a PR-B-dependent manner, and it is involved in cell cycle inhibition, apoptosis, and inhibition of migratory and invasive capacities in endometrial cancer [40][41][42][43] . Although the crosstalk between FOXO1 and PR is not strictly ligand-dependent, this crosstalk is important for decidualization as well as the induction of apoptosis 44,45 . Additionally, genistein has been reported to decrease the phosphorylation of Akt and increase the phosphorylation of p42/44 (ERK) in endometrial cancer cell lines 15 . Our data suggest that genistein inhibits cell proliferation by inducing the expression of FOXO1, which lies in the downstream of PI3K/PKB signaling pathway, regardless of ER status in endometrial cancer. Second, our results showed that genistein significantly increases the expressions of PR-AB and PR-B after 24 h of treatment, regardless of the ER status of the cell lines, and the effect was sustained up to 144 h in Ishikawa cells and 216 h in KLE cells. In fact, the C/EBPβ and the JNK pathway participated in the genistein-induced elevation of PR expression, rather than the estrogen responsiveness or epigenetic modification. One of the mechanisms for regulating the PR gene is by mediating estrogen responsiveness, even though the PR gene lacks a palindromic ERE sequence 6 . The promoter of PR-A contains a half ERE site upstream of two adjacent Sp1 sites, and the ER and Sp1 may play a role in activating the PR-A promoter 10,46 . However, our study suggests that the genisteinstimulated PR expression is not ER-mediated because genistein hardly changed the mRNA levels of ERα and ERβ; moreover, it decreased the protein level of ERα in the Ishikawa cells. Epigenetic modification of the PR promoter has been hypothesized to be another mechanism for regulating PR expression [47][48][49][50][51] . According to a study investigating the methylation status and the expressions of the two PR isoforms in endometrial cancer samples, the specimens with methylated PR-B alleles were negative for the immunohistochemical expression of PR-B, but all of them had unmethylated PR-A alleles 47  www.nature.com/scientificreports/ lines, HEC-1B and KLE, were highly methylated, and treatment with 5-aza-2ʹ-deoxycytidine, a DNA methyltransferase inhibitor, led to an increase in PR-B mRNA along with demethylation of the PR-B promoter in both the cell lines 47,48 . On the contrary, our MSP results indicated that the PR-B promoter was unmethylated prior to the genistein treatment, and the PR-A promoter remained methylated post-treatment in both the cell lines. Thereafter, we focused on c-Jun and c-Fos, which had been listed as candidates for transcription factors using JASPAR, since the mitogen-activated protein kinase (MAPK) pathway and Ras activation have been reported as mechanisms for regulating PR expression [52][53][54][55] . A previous report states that C/EBPβ and c-Jun synergize to stimulate a PR promoter-reporter, thereby elevating endogenous PR expression in murine mammary gland 17 , while another report states that C/EBPβ negatively regulates PR expression in human glioblastoma cells 16 . Our results show that genistein lowers C/EBPβ expression, elevates c-Jun expression, and increases phosphorylation of c-Jun and JNK. Genistein has always been considered as a non-specific protein-tyrosine kinase inhibitor 56 . Hence, genistein can maintain long-term PR-inducing effects as a tyrosine kinase inhibitor, regardless of ER status in endometrial cancer. www.nature.com/scientificreports/ Finally, the long-term PR-inducing effect of genistein can lead to an improved prognosis in patients with endometrial cancer. Since genistein-induced expression of PR and FOXO1 induces endometrial decidualization, it may be useful for maintenance treatment in young patients with endometrial cancer who wish to preserve their fertility. It has been reported that metformin also promotes PR expression via inhibition of the mammalian target of rapamycin in endometrial cancer cells 57 . A clinical trial has already shown that the combination of medroxyprogesterone and metformin is effective in terms of relapse-free survival and post-treatment conception 58 . Genistein has been found to be safe and non-teratogenic when taken via diet 59 . Therefore, genistein is expected to have an effect similar to metformin in young patients with endometrial cancer.
This study had several limitations. One of the limitations is determining the dosage of genistein intake required to increase PR expression and suppress cell proliferation. In vivo, the growth of melanoma tumors implanted into mice was inhibited by 50% when the mice were fed on a genistein-rich diet (1 mg/day). Plasma genistein concentration at the time of tumor removal was 1.1 μM 60 . This is similar to the levels reported in humans who were given two different preparations of unconjugated soy isoflavones, 4.3 and 16.3 μM 61 , but higher than the mean plasma levels detected in Japanese men consuming diets with a high content of soy products 62 . It is difficult to directly compare concentrations used in vitro or in vivo with circulating concentrations in humans. Epidemiological studies have shown that high genistein intake reduces the frequency of endometrial cancer 63 . Thirteen revised epidemiological studies revealed that isoflavone intake from soy products and legumes is associated with a 19% reduction in endometrial cancer risk 64 . Another limitation is the consideration of stromal PR expression in the tumor microenvironment. In the endometria of premenopausal women, both PR-A and PR-B isoforms are expressed in the epithelial and stromal cells 65 , and both isoforms appear to fluctuate in the cycling endometrium in a cell-specific manner 7 .
In conclusion, our study revealed that genistein induces long-time PR expression and inhibits cell proliferation in an ER-independent manner. Genistein may be a promising therapeutic candidate for improving the prognosis of young patients with endometrial cancer who wish to preserve their fertility.

Methods
Patients, specimen collection, and immunohistochemical analysis. Specimens collected from 32 uterine endometrial cancer patients aged < 40 years were evaluated. These patients had undergone dilatation and curettage or hysterectomy at the Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine (Kyoto, Japan) between 1997 and 2019. Among them, six patients who had undergone hysterectomy due to ineffective medroxyprogesterone acetate therapy were included. The research protocol was approved by the Institutional Review Board (ERB-C-2412) and performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all the patients prior to the study. Immunohistochemical staining was performed, as previously described 66,67 . Furthermore, the level of PR expression was assessed by H-score, a commonly used method to measure the strength of ER-and PR-staining that evaluates the intensity of staining and the percentage of cells stained at each intensity in a semi-quantitative manner. Intensities were scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The H-score was calculated using the following algorithm: H-score = ∑ (i + 1) × Pi (where i and Pi represent the intensity of staining and percentage of cells at each intensity, respectively). We used the quantification method to analyze the relation of H-score to survival rate, thereby categorizing the H-score into 2 groups, namely 1-100 and > 100. . We used the ATCC Ⓡ human short-tandem repeat (STR) profiling cell authentication service to confirm that our Ishikawa cell line sample was a match to the STR profile for this cell line listed on the ExPASy website, and our KLE cell line sample was an exact match to the ATCC cell line CRL-1622 (KLE). The Ishikawa cells were maintained in Minimum Essential Medium (MEM; Nacalai Tesque, Kyoto, Japan) with sodium pyruvate, and the KLE cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (Nacalai Tesque). Each medium was supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaille, France) and penicillin-streptomycin (Nacalai Tesque). All cells were cultured at 37 °C in a humidified atmosphere 5% CO 2 .
Flow cytometry analysis. Cells were seeded in 6-well plates and incubated for 7 days while changing the medium every 2 days. Cells were permeabilized with 0.1% Triton-X100 and the nuclei were then stained with propidium iodide (PI). The DNA content was measured using a fluorescence-activated cell sorting (FACS) Caliber cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with ModFit LT (Verty Software, Topsham, ME, USA) and Cell Quest software packages (BD Biosciences).
DNA extraction and sodium bisulfite sequencing. Genomic DNA was extracted from the cultured cells using DNAiso Reagent (Takara Bio Inc., Shiga, Japan), and 10 ng of the isolated DNA was subjected to sodium bisulfite conversion using EpiTect Ⓡ Fast DNA Bisulfite Kit (QIAGEN), according to the manufacturer's instructions. The converted DNA was eluted from DNA affinity columns, and 2 μL was used for subsequent PCR analyses.

The MSP analysis. The PCR analysis was performed using Premix Ex Taq™ Hot Start Version (Takara Bio
Inc.) and a hot start procedure. The following primers were used for this process: PR Animal model. Female BALB/c nu/nu mice (4-weeks-old) were purchased from Shimizu Co., Ltd. (Kyoto, Japan) and housed under specific pathogen-free conditions, as previously reported. Ishikawa cells (8.5 × 10 6 cells per mouse) were inoculated subcutaneously into the flanks of the mice (8-weeks-old). The resulting tumor volumes were calculated using the formula: 1/2 × (length) × (width) 2 . After establishment of a palpable tumor (approximately 30 mm 3 ), the mice were randomly divided into control and genistein groups (n = 6 for each). Subsequently, for 4 weeks, the mice in the genistein group received abdominal injections of genistein (90 mg/kg body weight) dissolved in DMSO on alternate days. Mice in the control group were treated with an equal volume of the vehicle. Tumor volume and body weight were sequentially measured once every 2 or 3 days for 4 weeks.